Follicular B helper T (Tfh) cells are required for normal immune responses, promoting development of memory B cells and long-lived plasma cells. When aberrantly regulated, such as in systemic lupus erythematosus (SLE, lupus), they drive maturation of autoreactive memory B cell and pathogenic plasma cell formation. Modulation of T-B cell interactions in murine models of lupus ameliorates disease, with promise that such intervention will be therapeutically beneficial in human lupus. Thus, it is reasonable to develop a more comprehensive understanding of the mechanisms that govern Tfh cell differentiation, survival, and collaboration with B cells in normal and disease settings. Upon activation, CD4 T cells exhibit dynamic changes in metabolism to meet their proliferative and effector needs. The understanding of how metabolism is regulated in Tfh cells is currently insufficient, although recent work has shown that interference with T cell metabolic programming is as beneficial in lupus models as it is in cancer immunotherapy, albeit without identification to-date of the specific T cell target(s). As Tfh cells operate in the unique GC niche in comparison to their T helper effector counterparts, we hypothesize that these cells utilize different programs of metabolism to fuel their function, with targeting such nodes proposed as a strategy for reprogramming cytotoxicity in tumor-infiltrating T lymphocytes. In the first, R61 phase of this project, we will establish in vivo, high-throughput sgRNA library screening system that supports Tfh cell generation from adoptively transferred T cells, transduced with a metabolome sgRNA library which allows for sufficient library coverage; provides enough sensitivity such that Cas9-mediated genetic lesions lead to observable Tfh cell phenotypes; and permits tracking of individual viral integration events. To achieve these goals, we will take advantage of an acute Armstrong LCMV (lymphocytic choriomeningitis virus) infection model we have used to interrogate Tfh cell differentiation, in which we can track development and differentiation of adoptively transferred T cells genetically manipulated with shRNA expressing retroviruses. This model will be adapted to our retroviral sgRNA system. We will use sgRNA against genes known to critically regulate Tfh cell development to establish benchmarks against which to evaluate the pooled screen and to define the dynamic range of the experimental setup. We also will generate a new barcoded vector to track individually transduced T cells, which will both enhance resolution of the screen and provide robust statistical power for the analysis. Once these goals have been achieved, we will proceed to generating the barcoded sgRNA library and conducting the in vivo screen. In the second, R33 phase, we will generate novel knockout animal models using using Cas9 technology to validate relevance of screen hits, with verified targets bred to lupus-prone mice in order to directly test the role of candidate metabolic genes in autoimmune disease.